Ricardo Wagner de Almeida Vitor

Congenital Toxoplasmosis in Southeastern Brazil: Results of Early Ophthalmologic Examination of a Large Cohort of Neonates

OBJECTIVE To report results of early ophthalmologic examinations in a large cohort of newborns with congenital toxoplasmosis (CT) after neonatal screening. DESIGN Cross-sectional analysis of a cohort. PARTICIPANTS A total of 178 newborns with confirmed CT from 146,307 screened babies (95% of live births) from Minas Gerais state, southeastern Brazil. METHODS From November 2006 to May 2007, newborns underwent neonatal screening by immunoglobulin (Ig)M capture of dried blood samples. On all positive or suspected cases, confirmative serology was performed on babies and their mothers. Congenital toxoplasmosis was confirmed in newborns who had IgM and/or IgA and IgG, or IgG associated with suggestive ocular lesions (with IgM and IgG in the mother). Ophthalmologic evaluation consisted of indirect ophthalmoscopy with a lid speculum. Pediatric examination and radiologic studies of the central nervous system were also performed. In selected cases, biomicroscopy of the anterior segment, fundus photographs, or ultrasonography (B-scan) was performed. MAIN OUTCOME MEASURES Prevalence of retinochoroidal lesions, either cicatricial or active, and their location and associated findings, such as vascular sheathing, hemorrhage, vitreous opacities, and retinal detachment, were evaluated. The occurrence of cataract, microphthalmia, microcephaly, intracranial calcification, and hydrocephalus was also recorded. RESULTS Of 146,307 neonates screened, 190 had CT, yielding a prevalence of 1 in 770 live births, of whom 178 (93.7%) underwent standardized ophthalmologic examination at an average age of 55.6+/-16.6 days. Of these 178 infants, 142 (79.8%) had retinochoroidal lesions consistent with CT in at least 1 eye. Bilateral involvement was noted in 113 patients (63.5%). Macular involvement was seen in 165 eyes (46.3%) of 111 patients (62.4%). Active lesions were observed in 142 eyes (39.9%) of 85 patients (47.8%). These lesions were located in the macula of 75 eyes (21.1%) and were associated with retinal vascular sheathing in 44 eyes (12.4%). CONCLUSIONS A high prevalence of CT was encountered (1/770) with high rates of early retinochoroidal involvement ( approximately 80%) and many active lesions (in approximately 50%), indicating a possibly more severe ocular involvement by CT in Brazil than in other parts of the world. The hypotheses of higher parasite virulence and increased individual susceptibility are being currently investigated.

Genetic Characterization of Toxoplasma gondii Revealed Highly Diverse Genotypes for Isolates from Newborns with Congenital Toxoplasmosis in Southeastern Brazil

ABSTRACT Recent studies of Toxoplasma gondii isolates from animals in Brazil have revealed high genetic diversity. Many of these isolates are virulent to mice. It is speculated that these isolates may also be virulent to humans. However, there is very limited data regarding T. gondii strains from human infection. Therefore, it is not clear whether there is any association between parasite genotypes and disease phenotypes. In this study, a total of 27 T. gondii strains were isolated from humans with congenital toxoplasmosis in Minas Gerais state, Brazil. The genetic variability was assessed by restricted fragment length polymorphism in 11 loci (SAG1, 5′ plus 3′ SAG2, alternative [alt.] SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). Genetic analysis of 24 strains revealed 14 different genotypes, including 7 previously identified from animals and 7 new types. The widespread genotype BrII accounted for 29% (7/24) of the isolates and was the dominant genotype involved in this study. This is the first report of genotyping of T. gondii isolates obtained from blood samples from newborns with congenital toxoplasmosis. Genotypic characterization of these isolates suggests high genetic diversity of T. gondii in this human population in Brazil. Future studies are needed to determine the source of contamination of this human population.

Role of Cytokines and Major Histocompatibility Complex Restriction in Mouse Resistance to Infection with a Natural Recombinant Strain (Type I-III) of Toxoplasma gondii

ABSTRACT Herein we characterized various genetic markers and the biological behavior of a natural recombinant strain of Toxoplasma gondii (P-Br). From nine genetic markers analyzed, three (B1, ROP1, and SAG1) and three (cS10-A6, GRA6, and SAG3) markers belong to parasites from the type I and type III lineages, respectively. The SAG2 and L363 loci were shown to be type I-III chimera alleles. The cB2l-4 microsatellite marker showed a unique haplotype. The P-Br strain presented low virulence in the acute phase of infection and was cystogenic during the chronic infection. The interleukin 12/gamma interferon axis and inducible nitric oxide synthase were main determinants of resistance during the acute infection with the P-Br strain. As opposed to infection with the type II strain of T. gondii (ME-49), peroral infection with the P-Br strain led only to a light inflammatory infiltrate and no major lesions in the intestine of the C57BL/6 mice. In addition, the BALB/c (resistant to ME-49) and C57BL/6 (susceptible to ME-49) mice were shown, respectively, to be more susceptible and more resistant to cyst formation and toxoplasmic encephalitis when infected with the P-Br strain. Further, the C57BL/KsJ and DBA2/J congenic strains containing major histocompatibility complex (MHC) haplotype “d” were more resistant than the parental strains (C57BL/6 and DBA1/J), when infected with the ME-49 but not with the P-Br strain. Together, our results indicate that resistance to cyst formation and toxoplasmic encephalitis induced during infection with P-Br is not primarily controlled by the MHC haplotype d, as previously reported for type II strains of T. gondii.

A Multihousehold Study Reveals a Positive Correlation between Age, Severity of Ocular Toxoplasmosis, and Levels of Glycoinositolphospholipid-Specific Immunoglobulin A

A cross-sectional household study involving 499 individuals was undertaken in an area of Minas Gerais state, Brazil, where infection with Toxoplasma gondii is endemic. Nearly 50% (n=247) of the sample had T. gondii-specific antibodies, even individuals in the 5-9-year-old age group. Approximately 12.5% (n=28) of a random subsample of participants who were positive for T. gondii antibodies had ocular lesions associated with T. gondii infection. The frequency of ocular toxoplasmosis increased significantly with age, with approximately 50% of individuals >60 years of age having lesions. The size of the ocular lesion correlated positively (r=0.85; P=.01) with the serum level of immunoglobulin A specific for tachyzoite-derived glycoinositolphospholipids. We found that sharing the same residence accounted for 30% of the variation in infectivity among residents in the sample, whereas age was the main risk factor for development of ocular toxoplasmosis in patients who were positive for T. gondii antibodies.

Risk factors for infection by Toxoplasma gondii in herds of goats in Ceará, Brazil

In order to identify possible risk factors for T. gondii infection in goat herds in Ceara, Brazil, 2362 serum samples were tested by ELISA. The serological prevalence was 25.1%. The risk factors identified for Toxoplasma gondii infection in goat herds were age, number of cats, use of wooden feeding troughs and absence of feeding troughs. Goats older than 37 months had 2.01 (CI 95%; 1.55 - 2.61) higher risk of infection than younger animals. Greater risk of infection was observed in farms with more than 10 cats (OR = 1.73; CI 95%; 1.01 - 3.33). The use of wooden feeding troughs represented a high probability of infection (OR = 7.81; CI 95%; 1.66 - 36.67). The lack of feeding troughs also represented a high probability of infection (OR = 5.50; CI 95%; 1.24 - 24.39).

Prenatal toxoplasmosis diagnosis from amniotic fluid by PCR

Toxoplasmosis is one of the most common infections all over the world. Most cases are asymptomatic, except in immunosuppressed individuals and fetuses, which can be seriously damaged. Prenatal diagnosis should be made as soon as possible since treatment of the mother can minimize fetal sequelae. Our aim in this study was to test the polymerase chain reaction technique (PCR) in 86 samples of amniotic fluid from women who seroconverted during pregnancy. DNA was amplified using external primers and, in a second step, internal primers, in a nested PCR system. Samples were also inoculated into mice and the newborn were evaluated by T. gondii serology, skull x-ray, transfontanel ultrasound, fundoscopic examination, lumbar puncture and clinical examination. PCR was positive in seven cases and negative in 79. Among PCR-positive cases, two were negative by inoculation into mice and by clinical evaluation; among PCR-negative ones, three had clinical evidence of toxoplasmosis and one was positive after inoculation into mice. PCR showed values of sensitivity = 62.5% and specificity = 97.4%; the values of inoculation into mice where 42.9% and 100%, respectively. Although PCR should not be used alone for prenatal diagnosis of congenital toxoplasmosis, it is a promising method and deserves more studies to improve its efficacy.

Overlapping Toxoplasma gondii genotypes circulating in domestic animals and humans in Southeastern Brazil.

Although several Toxoplasma gondii genotyping studies have been performed in Brazil, studies of isolates from animals in the state of Minas Gerais are rare. The objective of this study was to conduct a genotypic characterization of T. gondii isolates obtained from dogs, free-range chickens, and humans in Minas Gerais and to verify whether the T. gondii genotypes circulating in domestic animals correspond to the genotypes detected in humans. Genetic variability was assessed by restricted fragment length polymorphism at 11 loci (SAG1, 5′+3′SAG2, SAG2 alt, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). Twelve different genotypes were identified among the 24 isolates studied, including 8 previously identified genotypes and 4 new genotypes. The genetic relationship of the 24 T. gondii isolates, together with the genotypes previously described from 24 human newborns with congenital toxoplasmosis, revealed a high degree of similarity among the genotypes circulating in humans and animals in Minas Gerais. The most common genotypes among these species were BrII, BrIII, ToxoDB #108, and ToxoDB #206. Restricted fragment length polymorphism at the CS3 locus of these 48 isolates showed that the majority of isolates presented alleles I (50%) or II (27%). Isolates harboring allele III at the CS3 locus presented low virulence for mice, whereas those harboring alleles I or II presented higher virulence. These results confirm the utility of marker CS3 for predicting the virulence of Brazilian isolates of T. gondii in mice. No association was found between the allele type and clinical manifestations of human congenital toxoplasmosis. This is the first report of T. gondii genotyping that verifies the overlapping genotypes of T. gondii from humans and animals in the same geographic region of Brazil. Our results suggest that there is a common source of infection to the species studied, most likely oocysts contaminating the environment.

Vertical toxoplasmosis in a murine model. Protection after immunization with antigens of Toxoplasma gondii incorporated into liposomes

Distinct Toxoplasma gondii antigens were entrapped within liposomes and evaluated for their ability to protect Balb/c mice against congenital transmission: soluble tachyzoite antigen (L/STAg), soluble tissue cyst antigen (L/SCAg), soluble tachyzoite plus tissue cyst (L/STCAg) or purified 32kDa antigen of tachyzoite (L/pTAg). Soluble tachyzoite antigen alone in PBS (STAg) or emulsified in Freunds Complete Adjuvant (FCA/STAg) was also evaluated. Dams were inoculated subcutaneously with these antigens 6, 4 and 2 weeks prior to a challenge with four tissue cysts of the P strain of T. gondii orally between 10 and 14 days of pregnancy. Significant diminution differences were observed between the frequency of infected pups born of the dams immunized with the antigens incorporated into liposomes and that of pups born of the dams immunized with antigen emulsified in FCA or non immunized group (p<0.05). There was a significant decrease in the number of pups born dead in the groups L/STAg, L/SCAg and L/pTAg when compared with pups from all other groups (p <0.05). All dams immunized with or without adjuvant showed an antibody response and a proliferation of T-cells. However, no correlation was found between immune response and protection against the challenge.

Congenital toxoplasmosis from a chronically infected woman with reactivation of retinochoroiditis during pregnancy.

OBJECTIVE To report a rare case of congenital toxoplasmosis from an immunocompetent mother with chronic infection who had reactivation of ocular disease during pregnancy. DESCRIPTION The newborn was asymptomatic at birth and identified by neonatal screening (IgM anti-Toxoplasma gondii in dried blood) among other 190 infants with congenital toxoplasmosis during a 7-month period. His mother had had a non-treated episode of reactivation of toxoplasmic retinochoroiditis during pregnancy, with stable IgG titers and negative IgM results. Results of IgM and IgG in the newborns serum, as well as IgG immunoblotting were positive and active retinochoroidal lesions were detected in his peripheral retina. The neonate was treated with sulfadiazine, pyrimethamine and folinic acid. At 14 months of life, the child remained asymptomatic, with regression of retinochoroidal lesions and persistence of IgG. COMMENTS It is possible that systematic neonatal screening in areas with high prevalence of infection may identify these cases.

Western blot analysis of the humoral response of dogs experimentally infected with Angiostrongylus vasorum (Baillet, 1866)

Seven cross-bred dogs were inoculated with Angiostrongylus vasorum and serum samples were analyzed using the enzyme-linked immunosorbent assay (ELISA) and Western blot (WB). ELISA detected specific antibodies anti-A. vasorum, from 14 to 28 days after inoculation (DAI) and persisted throughout the experiment. Using WB, the main antigens detected had molecular weight of approximately 115, 102, 86, 76, 69, 56, 41, 32, 28, 20-22 and 10kDa.